This invention relates to a process for measuring complement activity by quantitative analysis and a reagent composition used therefor.
The complement system is a collective term of about 20 kinds of proteins present in animal serum such as human serum. The complement system plays an important role for defending living bodies from invading foreign substances and bacteria by recognition and removal thereof. It is well known that the complement system has two pathways for activation, that is, the classical pathway wherein the activation is brought about by an immune complex and the alternative pathway wherein the activation is brought about by polysaccharides. In the classical pathway, the complement component is orderly activated one after another by an immune complex of a foreign substance (antigen) such as bacterium mainly, and an antibody to the antigen to finally destroy the foreign substance membrane, resulting in making the foreign substance die or lyse. The classical pathway includes, for example, a reaction wherein erythrocyte sensitized by hemolysin is led to hemolysis, and a reaction wherein liposome membrane sensitizing hapten thereon is destroyed as in the present invention. On the other hand, the alternative pathway does not require participation of an antibody. For example, in the alternative pathway, complement is activated by only a contact with a polysaccharide, which is a component constituting cell walls of bacteria, or a virus. Recently, the measurement of activity of a complement system, that is, the measurement of a complement activity has been noticed as an index of diagnosis and therapy of active stage of systemic lupus erythematosus, rheumatoid arthritis, cryoglobulinemiavasculitis, some forms of nephritis, and inherited deficiencies of the complement system etc.
As a process for measuring a complement activity now generally and widely used, it is known the Mayer method or its variations wherein hemolysis activity of complement is measured using antibody-sensitized sheep erythrocyte ["Rinsho Kensaho Teiyo", pp. 1233-1234, 29ed, 5th print, 1985 published by Kanehara Shuppan Co., Ltd.; J. Clin. Chem., 12, 143 (1983), etc.]. But the Mayer method is a very complicated method wherein a number of samples with various dilutions for one sample to be measured are prepared to carry out the reaction, and complement activity is measured by obtaining an amount for bringing about 50% hemolysis (CH.sub.50 U/ml) based on such results. Further, there are various problems in that the erythrocytes used are poor in storage stability, sensitivity for complement is different depending on individual differences of animals, etc. Therefore, the Mayer method cannot be said as a preferable method.
On the other hand, it is reported various methods for measuring complement activity using liposomes which are more stable, less different between production lots, and prepared so as to be subjected to membrane damaging reaction by complement activity in place of erythrocytes (Japanese Patent Unexamined Publication (JP-A) Nos. 63-293470, 1-155271, 62-16396, 62-299764, etc.). But even in these methods using liposomes, there is a problem of sedimentation and aggregation of the liposomes during storage of reagent solutions dispersing the liposomes. Particularly, in the measurement using an autoanalyzer, such a phenomenon causes various problems. For example, since almost autoanalyzers are designed to have a cooling device for reagent solutions, which can be subjected to the measurement at any time while set in the autoanalyzer, when sedimentation and aggregation in the reagent solutions take place during the storage or the long-period measurement, the measurement is damaged due to variation of measuring sensitivity.
In order to solve such problems, it is proposed to use only liposomes immobilizing a specific functional group such as a pyridyl group in place of using the liposomes immobilizing an antibody thereon and an antigen to the antibody. But since such a method has a problem in that the reaction principle is different from the Mayer method wherein complement activity is measured based on the antigen-antibody reaction, this method is not preferable. Further, since these prior art measuring methods usually require the measurement at 37.degree. C. for a reaction time of 1 hour, there is another problem in that the reaction time is too long to apply them to autoanalyzers.
Recently, the measurement using an autoanalyzer is particularly noticed and the measurement of human complement activity using an autoanalyzer is earnestly desired. But, since usual autoanalyzers are designed so as to conduct reactions for about 5 to 15 minutes, it is difficult to apply human complement activity measurement requiring a long period of reaction time to such autoanalyzers.
In addition, at the time of measuring human complement activity, calibration curves are usually prepared by using complement activity standard solutions, and the complement activity in samples are generally obtained by using the calibration curves. As the standard solutions for complement activity, there are generally used human serum and guinea pig serum. Since the human serum is difficult to obtain and difficult to control, commercially available reagents for the measurement often attaching guinea pig serum as standard solutions for complement activity. But, there is a difference between human complement and guinea pig complement in the reaction rate at the initial time of the reaction [J. Immunol. Methods, 17, 7-19 (1977)]. Therefore, when guinea pig complement is used as standard solutions for measuring human complement activity, since a long time is required for the measurement, there is a problem for applying said method to autoanalyzers.
Considering the above-mentioned problems, it has long been desired to develop methods for measuring human complement activity using liposomes stabler than erythrocytes derived from living bodies and less in difference between production lots, with simpler procedures and shorter time and being able to apply to autoanalyzers, and to develop reagent compositions for the measurements.